S138: MICROENVIRONMENT-REGULATED TRANSCRIPTIONS FACTORS AHR AND HIF-1Α EXPRESSION IN TREGS PROMOTE CLL PROGRESSION BY IMPAIRING CD8+ T-CELL MEDIATED ANTI-TUMOR IMMUNITY
Authors: G. Pagano, M. Wierz, I. Fernandez Botana, S. Gonder, E. Gargiulo, E. Moussay, J. Paggetti
Journal: HemaSphere (2022)

Background: Chronic Lymphocytic Leukemia (CLL) progression is highly dependent on complex interactions between tumor cells and the tumor microenvironment (TME). Indeed, CLL cells can modify stromal cells and immune cells to promote the survival of the leukemic clone and to escape from the immune system surveillance. Within the TME, regulatory T cells (Tregs) represent a subtype of CD4+ T cells with immunosuppressive abilities, causing the evasion of cancer cells from the immune system. We previously characterized extensively the immune microenvironment of pre-clinical CLL mouse models using mass cytometry, and we described a significant increase in the Tregs subsets with an enhanced immunosuppressive and activated phenotype compared to non-leukemic animals (Wierz et al., Blood, 2018). Interestingly, TIGIT+ Tregs are more immunosuppressive than their TIGIT- Treg counterparts and express higher levels of several transcription factors, including Ahr and Hif1α (Joller et al., Immunity, 2014), both involved in the cellular response to microenvironment-mediated stimuli. Aims: The aim of the present study is to investigate the role of AHR and HIF-1α in the suppressive ability of Tregs during CLL development. Methods: We generated conditional knock out mice (cKO) lacking Ahr or Hif1a genes exclusively in Tregs (Foxp3YFP-CreAhrfx/fx and Foxp3YFP-CreHif1afx/fx mice). We then performed adoptive transfer (AT) of CLL cells obtained from diseased Eµ-TCL1 mice into cKO and control mice. In order to decipher the mechanism by which AHR and HIF-1α pathways in Tregs affect CLL progression, we analyzed the splenic TME of recipient mice and evaluated immune checkpoint expression and cytokine production. Finally, we evaluated the suppression ability of the regulatory T cells with ex vivo suppression assays. Results: Generated cKO mice showed no sign of abnormalities or autoimmune phenotypes, and immune cells phenotyping revealed no major differences. However, we showed that CLL growth in cKO mice was drastically delayed compared to the control mice (A). Interestingly, this decrease was mitigated when CD8+ T cells were depleted. The analysis of the splenic TME in recipient cKO mice revealed an increase in IL-17 and TNF-α production, two major T-cell cytokines, in CD4+ T cells as compared to their WT counterparts. We also measured the expression of immune checkpoints and activation markers in the different T cell subpopulations and observed that Tregs lacking AHR or HIF-1α show decreased levels of the immune checkpoints CTLA-4 and TIGIT, two key proteins for the suppressive functions of Tregs. Ex vivo suppression assays demonstrated an increased proliferation of CD8+ T cells in the presence cKO Tregs (B-C). This result confirmed the decreased suppressive ability of Tregs in absence of the two transcription factors, explaining the observed delay in CLL progression in cKO mice. Image:Summary/Conclusion: Altogether, these results indicate that the TME-regulated transcription factors AHR and HIF-1α in Tregs are crucial for CLL development by promoting escape to anti-tumor immune response, and therefore represent potential therapeutic targets during CLL progression.